一株海洋低温葡萄糖氧化酶担子菌的诱变育种及其酶学性质研究
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摘要
利用紫外诱变、化学诱变及紫外-化学复合诱变技术对一株海洋来源的产低温葡萄糖氧化酶(GOD)担子菌(Basidioascus sp.)WYQ 23进行诱变育种,并对其遗传稳定性及所产酶酶学性质进行研究。结果表明,获得一株高产低温葡萄糖氧化酶的突变菌株Basidioascus sp. WYQ 23-3-4。该突变菌株GOD酶活为2.33 U/m L,是原始菌株的1.40倍。经8代传代培养实验表明,突变菌株WYQ23-3-4产葡萄糖氧化酶能力稳定。该酶最适温度为20℃,在10～30℃可保持较高酶活;最适pH值为5.6,在p H5.0～6.0可保持较高酶活;金属离子Ag~+、Zn~(2+)、Fe~(2+)对酶活抑制作用较强,Mg~(2+)、K~+对酶有激活作用。综上,通过复合诱变可明显提高担子菌产低温葡萄糖氧化酶的能力,为该酶的工业化生产提供了潜在的优良的菌种资源。
A low temperature glucose oxidase(GOD)-producing Basidioascus sp. WYQ 23 from marine was screened by ultraviolet mutagenesis,chemical mutagenesis and ultraviolet-chemical compound mutagenesis, and its genetic stability and enzymatic properties were studied. The results showed that a mutant strain Basidioascus sp. WYQ 23-3-4 with high yield of low temperature glucose oxidase was obtained. The GOD activity of the mutant strain was 2.33 U/ml, which was 1.40 times higher than that of the original strain. After 8 generations of culture, the mutant strain WYQ 23-3-4 had stable ability to produce glucose oxidase. The optimum temperature of the enzyme was 20 ℃, and the enzyme could maintain the higher enzyme activity at 10-30 ℃. The optimum pH was 5.6, and the enzyme could maintain the higher enzyme activity at 5.0-6.0. Ag~+, Zn~(2+), Fe~(2+) had strong inhibitory effect on the enzyme activity, while Mg~(2+), K+had activation effect on the enzyme. In conclusion, the ability of Basidioascus sp. to produce low-temperature glucose oxidase could be significantly improved by compound mutagenesis, which provided potential excellent strain resources for the industrialization production of the enzyme.
A low temperature glucose oxidase(GOD)-producing Basidioascus sp. WYQ 23 from marine was screened by ultraviolet mutagenesis,chemical mutagenesis and ultraviolet-chemical compound mutagenesis, and its genetic stability and enzymatic properties were studied. The results showed that a mutant strain Basidioascus sp. WYQ 23-3-4 with high yield of low temperature glucose oxidase was obtained. The GOD activity of the mutant strain was 2.33 U/ml, which was 1.40 times higher than that of the original strain. After 8 generations of culture, the mutant strain WYQ 23-3-4 had stable ability to produce glucose oxidase. The optimum temperature of the enzyme was 20 ℃, and the enzyme could maintain the higher enzyme activity at 10-30 ℃. The optimum pH was 5.6, and the enzyme could maintain the higher enzyme activity at 5.0-6.0. Ag~+, Zn~(2+), Fe~(2+) had strong inhibitory effect on the enzyme activity, while Mg~(2+), K+had activation effect on the enzyme. In conclusion, the ability of Basidioascus sp. to produce low-temperature glucose oxidase could be significantly improved by compound mutagenesis, which provided potential excellent strain resources for the industrialization production of the enzyme.
引文
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[18]朱洪菊.葡萄糖氧化酶产生菌的诱变育种及条件优化[D].济南:山东师范大学,2012.
[19]张艳丽.葡萄糖氧化酶的高效表达及耐热性分子改良[D].北京:中国农业科学院,2018.
[20]郭云瑕,孙钰婷,辛颖,等.葡萄糖氧化酶产生菌的紫外诱变及发酵条件优化[J].食品科技,2016,41(5):2-6.
[21]聂金梅,李阳源,刘金山,等.黑曲霉葡萄糖氧化酶基因改造及其在毕赤酵母中的表达[J].江苏农业科学,2018,46(20):17-21.
[2]WANG Y Y,WANG Y B,XU H,et al.Direct-fed glucose oxidase and its combination with B.amyloliquefaciens SC06 on growth performance,meat quality,intestinal barrier,antioxidative status,and immunity of yellow-feathered broilers[J].Poul Sci,2018,97(10):3540-3549.
[3]TAHOUN M K.Intensification of glucose oxidase synthesis by multistage mutagenesis of Aspergillus niger[J].Appl Biochem Biotechnol,1993,39(1):289-295.
[4]李蓉,张庆芳,迟乃玉.微生物葡萄糖氧化酶的研究进展[J].中国酿造,2018,37(3):1-5.
[5]BANKEFA O E,WANG M Y,ZHU T C,et al.Enhancing the secretion pathway maximizes the effects of mixed feeding strategy for glucose oxidase production in the methylotrophic yeast Pichia pastoris[J].Bioresourc Bioproc,2018,5(1):25.
[6]廖兆民,蔡俊,林建国.微生物葡萄糖氧化酶的研究进展[J].食品与发酵工业,2018,44(7):308-315.
[7]石淑钰,张庆芳,迟乃玉,等.一株海洋低温葡萄糖氧化酶菌株的筛选、鉴定及部分酶学性质[J].微生物学通报,2014,41(5):832-838.
[8]黄晓月,莫日坚,徐德峰,等.海洋源Bacillus cereus CAMT2377产葡萄糖氧化酶过程分析及优化[J].中国生物制品学杂志,2018,31(11):1268-1273.
[9]FREDERICK K R,TUNG J,EMERICK R S,et al.Glucose oxidase from Aspergillus niger.Cloning,gene sequence,secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme[J].J Biol Chem,1990,265(7):3793-3802.
[10]徐军庆.葡萄糖氧化酶产生菌的诱变育种及条件优化[D].济南:齐鲁工业大学,2014.
[11]陈楠,肖成建,范新蕾,等.黑曲霉葡萄糖氧化酶基因在毕赤酵母SMD1168中的表达[J].食品与生物技术学报,2017,36(9):975-981.
[12]闻一凡,顾磊,张娟,等.定点突变提高毕赤酵母产葡萄糖氧化酶的氧化稳定性[J].食品与生物技术学报,2016,35(12):1260-1267.
[13]杨付伟.黑曲霉液体发酵产木聚糖酶的菌种选育和工艺研究[D].杭州:浙江大学,2018.
[14]应立立.高效利用玉米秸秆菌株的筛选及其对玉米秸秆的降解研究[D].吉林:吉林农业大学,2017.
[15]顾磊.Aspergillus niger葡萄糖氧化酶的异源分泌表达、分子改造和发酵生产[D].无锡:江南大学,2014.
[16]朱运平,褚文丹,黎芳,等.黑曲霉1504产葡萄糖氧化酶的ARTP诱变、产酶条件优化及改良面粉品质的研究[C].中国食品科学技术学会.中国食品科学技术学会第十三届年会论文摘要集:2016年.北京:中国食品科学技术学会学术学部:2016.
[17]刘晓筱.黑曲霉葡萄糖氧化酶在解脂耶氏酵母中的表达及酶学性质研究[D].无锡:江南大学,2016.
[18]朱洪菊.葡萄糖氧化酶产生菌的诱变育种及条件优化[D].济南:山东师范大学,2012.
[19]张艳丽.葡萄糖氧化酶的高效表达及耐热性分子改良[D].北京:中国农业科学院,2018.
[20]郭云瑕,孙钰婷,辛颖,等.葡萄糖氧化酶产生菌的紫外诱变及发酵条件优化[J].食品科技,2016,41(5):2-6.
[21]聂金梅,李阳源,刘金山,等.黑曲霉葡萄糖氧化酶基因改造及其在毕赤酵母中的表达[J].江苏农业科学,2018,46(20):17-21.